The goal of this SBIR is to develop a novel gene expression plasmid capable of long-term non- integrated episome maintenance with chemical tunable gene expression. The vector system can function in multiple human cell types, including rapidly dividing cancer cells, progenitor stem cells, or post-mitotic somatic cells. The major innovations are derived from the application of basic discoveries in genome maintenance and gene regulation from studies with human gamma herpes viruses. Specifically, we incorporate the use of chromatin insulator (INS) elements (consisting of binding sites for the cellular factor CTCF) to punctuate key regulatory features derived from Epstein-Barr Virus (EBV) and Kaposi's Sarcoma-Associated Herpes virus (KSHV). We incorporate the EBV chromosome architecture protein EBNA1 to maintain the episomal features and prevent epigenetic repression of transcription initiation sites at the EBV Q promoter (Qp). Components of the Qp prevent DNA methylation and epigenetic silencing, overcoming a major barrier to previous generations of gene expression vectors. In addition, we use a version of EBNA1 protein that limits immune recognition and MHC presentation. We also use the KSHV LANA promoter (LANAp) which functions efficiently in lymphoid and epithelial cells, and resists epigenetic suppression. Finally we propose to use newly developed small molecules regulators of EBNA1 to fine tune the episome maintenance and transcriptional control of transgenes cloned into the vector. As a proof of principle, we will express the cystic fibrosis gene CFTR in EBV plasmid system for long-term expression in human lung epithelial cells. This vector system will provide long-term regulatable expression of transgenes required for in vitro research and ultimately, human gene therapy. The vector can be further optimized for delivery in liposomes, nanoparticles, or modified viral like particles for more selective delivery in vivo. The proposed plasmid-expression system will have significant utility in human gene therapy.